Methods of assaying receptor activity and constructs useful in such methods

ABSTRACT

Described are methods of detecting G-protein coupled receptor (GPCR) activity in vivo and in vitro; methods of assaying GPCR activity; and methods of screening for GPCR ligands, G Protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process. Constructs useful in such methods are described.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NationalInstitutes of Health Grant No. HLO3422-02 and NS 19576. The Governmenthas certain rights to this invention.

This application is a continuation of U.S. Patent application Ser. No.08/869,568, filed Jun. 5, 1997, now issued as U.S. Pat. No. 5,891,646,the disclosure of which is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

This invention relates to methods of detecting G-protein coupledreceptor (GPCR) activity in vivo and in vitro, and provides methods ofassaying GPCR activity, and methods of screening for GPCR ligands, Gprotein-coupled receptor kinase (GRK) activity, and compounds thatinteract with components of the GPCR regulatory process. This inventionalso provides constructs useful in such methods.

BACKGROUND OF THE INVENTION

The actions of many extracellular signals are mediated by theinteraction of G-protein coupled receptors (GPCRs) and guaninenucleotide-binding regulatory proteins (G proteins). G protein-mediatedsignaling systems have been identified in many divergent organisms, suchas mammals and yeast. GPCRs respond to, among other extracellularsignals, neurotransmitters, hormones, odorants and light. GPCRs aresimilar and possess a number of highly conserved amino acids; the GPCRsare thought to represent a large `superfamily` of proteins. IndividualGPCR types activate a particular signal transduction pathway; at leastten different signal transduction pathways are known to be activated viaGPCRs. For example, the beta 2-adrenergic receptor (βAR) is a prototypemammalian GPCR. In response to agonist binding, βAR receptors activate aG protein (G_(s)) which in turn stimulates adenylate cyclase and cyclicadenosine monophosphate production in the cell.

It has been postulated that members of the GPCR superfamily desensitizevia a common mechanism involving G protein-coupled receptor kinase (GRK)phosphorylation followed by arrestin binding. Gurevich et al., J. Biol.Chem. 270:720 (1995); Ferguson et al., Can. J. Physiol. Pharmacol.74:1095 (1996). However, the localization and the source of the pool ofarrestin molecules targeted to receptors in response to agonistactivation was unknown. Moreover, except for a limited number ofreceptors, a common role for β-arrestin in GPCR desensitization had notbeen established. The role of β-arrestins in GPCR signal transductionwas postulated primarily due to the biochemical observations.

Many available therapeutic drugs in use today target GPCRs, as theymediate vital physiological responses, including vasodilation, heartrate, bronchodilation, endocrine secretion, and gut peristalsis. See,eg.., Lefkowitz et al., Ann. Rev. Biochem. 52:159 (1983). GPCRs includethe adrenergic receptors (alpha and beta); ligands to beta ARs are usedin the treatment of anaphylaxis, shock, hypertension, hypotension,asthma and other conditions. Additionally, spontaneous activation ofGPCRs occurs, where a GPCR cellular response is generated in the absenceof a ligand. Increased spontaneous activity can be decreased byantagonists of the GPCR (a process known as inverse agonism); suchmethods are therapeutically important where diseases cause an increasein spontaneous GPCR activity.

Efforts such as the Human Genome Project are identifying new GPCRs(`orphan` receptors) whose physiological roles and ligands are unknown.It is estimated that several thousand GPCRs exist in the human genome.With only about 10% of the human genome sequenced, 250 GPCRs have beenidentified; fewer than 150 have been associated with ligands.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a conjugate of a β-arrestinprotein and a detectable molecule. The detectable molecule may be anoptically detectable molecule, such as Green Fluorescent Protein.

A further aspect of the present invention is a nucleic acid constructcomprising an expression cassette. The construct includes, in the 5' to5' direction, a promoter and a nucleic acid segment operativelyassociated with the promoter, and the nucleic acid segment encodes aβ-arrestin protein and detectable molecule. The detectable molecule maybe an optically detectable molecule such as Green Fluorescent Protein.

A further aspect of the present invention is a host cell containing anucleic acid molecule which includes, a promoter operable in the hostcell and a nucleic acid sequence encoding a β-arrestin protein and adetectable molecule. The detectable molecule may be an opticallydetectable molecule such as Green Fluorescent Protein. The cell may be amammalian, bacterial, yeast, fungal, plant or animal cell, and may bedeposited on a substrate.

A further aspect of the present invention is a method of assessing Gprotein coupled receptor (GPCR) pathway activity under test conditions,by providing a test cell that expresses a GPCR and that contains aconjugate of a β-arrestin protein and a visually detectable molecule;exposing the test cell to a known GPCR agonist under test conditions;and then detecting translocation of the detectable molecule from thecytosol of the test cell to the membrane edge of the test cell.Translocation of the detectable molecule in the test cell indicatesactivation of the GPCR pathway. Exemplary test conditions include thepresence in the test cell of a test kinase and/or a test G-protein, orexposure of the test cell to a test ligand, or co-expression in the testcell of a second receptor.

A further aspect of the present invention is a method for screening aβ-arrestin protein (or fragment of a β-arrestin protein) for the abilityto bind to a phosphorylated GPCR. A cell is provided that expresses aGPCR and contains a conjugate of a test β-arrestin protein and avisually detectable molecule. The cell is exposed to a known GPCRagonist and then translocation of the detectable molecule from the cellcytosol to the cell edge is detected. Translocation of the detectablemolecule indicates that the β-arrestin molecule can bind tophosphorylated GPCR in the test cell.

A further aspect of the present invention is a method to screen a testcompound for G protein coupled receptor (GPCR) agonist activity. A testcell is provided that expresses a GPCR and contains a conjugate of aβ-arrestin protein and a visually detectable molecule. The cell isexposed to a test compound, and translocation of the detectable moleculefrom the cell cytosol to the membrane edge is detected. Movement of thedetectable molecule to the membrane edge after exposure of the cell tothe test compound indicates GPCR agonist activity of the test compound.The test cell may express a known GPCR or a variety of known GPCRs, orexpress an unknown GPCR or a variety of unknown GPCRs. The GPCR may be,for example, an odorant GPCR or a β-adrenergic GPCR. The test cell maybe a mammalian, bacterial, yeast, fungal, plan or animal cell.

A further aspect of the present invention is a method of screening asample solution for the presence of an agonist to a G protein coupledreceptor (GPCR). A test cell is provided that expresses a GPCR andcontains a conjugate of a β-arrestin protein and a visually detectablemolecule. The test cell is exposed to a sample solution, andtranslocation of the detectable molecule from the cell cytosol to themembrane edge is assessed. Movement of the detectable molecule to themembrane edge after exposure to the sample solution indicates the samplesolution contains an agonist for a GPCR expressed in the cell.

A further aspect of the present invention is a method of screening atest compound for G protein coupled receptor (GPCR) antagonist activity.A cell is provided that expresses a GPCR and contains a conjugate of aβ-arrestin protein and a visually detectable molecule. The cell isexposed to a test compound and to a GPCR agonist, and translocation ofthe detectable molecule from the cell cytosol to the membrane edge isdetected. When exposure to the agonist occurs at the same time as orsubsequent to exposure to the test compound, movement of the detectablemolecule from the cytosol to the membrane edge after exposure to thetest compound indicates that the test compound is not a GPCR antagonist.

A further aspect of the present invention is a method of screening atest compound for G protein coupled receptor (GPCR) antagonist activity.A test cell is provided that expresses a GPCR and contains a conjugateof a β-arrestin protein and a visually detectable molecule. The cell isexposed to a GPCR agonist so that translocation of the detectablemolecule from the cytosol of the cell to the membrane edge of the celloccurs, and the cell is then exposed to a test compound. Where exposureto the agonist occurs prior to exposure to the test compound, movementof the detectable molecule from the membrane edge of the cell to thecytosol after exposure of the cell to the test compound indicates thatthe test compound has GPCR antagonist activity.

A further aspect of the present invention is a method of screening acell for the presence of a G protein coupled receptor (GPCR). A testcell is provided that contains a conjugate of a β-arrestin protein and avisually detectable molecule. The test cell is exposed to a solutioncontaining a GPCR agonist. Any translocation of the detectable moleculefrom the cytosol to the membrane edge is detected; movement of thedetectable molecule from the cytosol to the membrane edge after exposureof the test cell to GPCR agonist indicates that the test cell contains aGPCR.

A further aspect of the present invention is a method of screening aplurality of cells for those cells which contain a G protein coupledreceptor (GPCR). A plurality of test cells containing a conjugate of aβ-arrestin protein and a visually detectable molecule are provided, andthe test cells are exposed to a known GPCR agonist. Cells in which thedetectable molecule is translocated from the cytosol to the membraneedge are identified or detected. Movement of the detectable molecule tothe membrane edge after exposure to a GPCR agonist indicates that thecell contains a GPCR responsive to that GPCR agonist. The plurality oftest cells may be contained in a tissue, an organ, or an intact animal.

A further aspect of the present invention is a substrate havingdeposited thereon a plurality of cells that express a GPCR and thatcontain a conjugate of a β-arrestin protein and a detectable molecule.Such substrates may be made of glass, plastic, ceramic, semiconductor,silica, fiber optic, diamond, biocompatible monomer, or biocompatiblepolymer materials.

A further aspect of the present invention is an apparatus fordetermining GPCR activity in a test cell. The apparatus includes meansfor measuring indicia of the intracellular distribution of a detectablemolecule, and a computer program product that includes a computerreadable storage medium having computer-readable program code meansembodied in the medium. The computer-readable program code meansincludes computer-readable program code means for determining whetherthe indicia of the distribution of the detectable molecule in a testcell indicates concentration of the detectable molecule at the cellmembrane, based on comparison to the measured indicia of theintracellular distribution of a detectable molecule in a control cell.The indicia of the intracellular distribution of the detectable moleculemay be optical indicia, and the measuring means may be means formeasuring fluorescent intensity. The molecule to be detected may be onethat is fluorescently detectable, and the step of measuring the indiciaof the intracellular distribution of the detectable molecule may includemeasurement of fluorescence signals from test and control cells.

A further aspect of the present invention is an apparatus fordetermining GPCR activity in a test cell. The apparatus includes meansfor measuring indicia of the intracellular distribution of a detectablemolecule in at least one test cell at multiple time points, and acomputer program product. The computer program product includes acomputer readable storage medium having computer-readable program codemeans embodied in said medium. The computer-readable program code meansincludes computer-readable program code means for determining whetherthe indicia of the distribution of the detectable molecule in the testcell at multiple time points indicates translocation of the detectablemolecule to the cell membrane.

A further aspect of the present invention is an apparatus fordetermining GPCR activity in a test cell, which includes means formeasuring indicia of the intracellular distribution of a detectablemolecule in at least one test cell, and a computer program product. Thecomputer program product includes a computer readable storage mediumhaving computer-readable program code means embodied therein andincluding computer-readable program code means for determining whetherthe indicia of the distribution of the detectable molecule in the testcell indicates concentration of the detectable molecule at the cellmembrane, based on comparison to pre-established criteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a linear model of the β-arrestin2/S65T-Green FluorescentProtein (GFP) conjugate.

FIG. 2A provides the results of a Western Blot of homogenates of HEK-293cells expressing the βarr2-GFP conjugate as well as endogenousβ-arrestin2-GFP conjugate; approximate molecular weights are indicatedto the right of the gel. Lane 1 was treated with anti-βarrestinantibody; Lane 2 with anti-GFP antibody.

FIG. 2B shows the sequestration of β2AR in COS cells with and withoutoverexpressed β-arrestin2 (left two bars) and with and withoutoverexpressed βarr2-GFP (right two bars). Wild type β-arrestin2 andβarr2-GFP enhanced β2AR sequestration equally well above control levels,producing a 2.5 and 2.4 fold increase, respectively.

FIG. 3A: Confocal microscopy photomicrographs show βarr2-GFPtranslocation from cytosol (panel 1 at left) to membrane (panel 2 atright) in HEK-293 cells containing the β2AR, due to the addition of theβAR2 agonist isoproterenol. Bar =10 microns.

FIG. 3B: Confocal microscopy photomicrographs show βarr2GFPtranslocation from cytosol (panel 1 at left) to membrane (panel 2 atright) in COS cells containing the β2AR, and due to addition of the βAR2agonist isoproterenol. Bar =10 microns.

FIG. 4 depicts a HEK-293 cell containing 12CA5(HA) tagged β2AR (confocalmicroscopic photographs). Row A shows a cell after reorganization ofβ2AR into plasma membrane clusters. Row B provides three pictures of thesame cell at 0, 3, and 10 minutes (left to right) after the addition ofagonist. Redistribution of βarr2-GFP to the cell membrane is shown bythe enhancement of membrane fluorescence with a concomitant loss ofcytosolic fluorescence. Arrows indicate areas of co-localization; bar=10microns.

FIG. 5 shows the influence of overexpressed GRK on the redistribution ofβarr2-GFP in HEK-293 cells expressing the Y326A phosphorylation-impairedβ2AR. Cells without (Row A) and with (Row B) overexpressed GRKs wereexposed to agonist, and the real-time redistribution of βarr2-GFP wasobserved. βarr2-GFP translocation in cells containing overexpressed GRK(Row B) was more robust, indicating an increased affinity of βarr2-GFPfor receptor. Bar =10 microns.

FIG. 6A depicts the agonist-induced time dependent translocation ofβarr2-GFP to beta2 adrenergic receptors in a representational HEK-293cell.

FIG. 6B graphs the time course of agonist-induced translocation ofβarr2-GFP to beta2 adrenergic receptors in HEK-293 cells; this graph isquantitative and is based on the responses of a plurality of cells.

FIG. 6C is depicts the agonist-induced translocation of βarr2-GFP tobeta2 adrenergic receptors in representational HEK-293 cells, at varyingdoses of agonist.

FIG. 6D graphs the dose dependent agonist-induced translocation ofβarr2-GFP to beta2 adrenergic receptors in HEK-293 cells; this graph isquantitative and is based on the responses of a plurality of cells.

FIG. 6E evaluates the translocation of βarr2-GFP from the cell cytosolto the cell membrane, in response to exposure to receptor agonist(middle panel) and subsequent exposure to receptor antagonist (rightpanel).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have determined that β-arrestin redistributionfrom the cytosol to the plasma membrane occurs in response to agonistactivation of GPCRs. The present inventors demonstrated a common rolefor β-arrestin in agonist-mediated signal transduction terminationfollowing agonist activation of receptors. The present inventors havedevised convenient methods of assaying agonist stimulation of GPCRS invivo and in vitro in real time. Although the pharmacology of members ofthe GPCR superfamily differs, the methods of the present inventionutilize β-arrestin translocation to provide a single-step, real-timeassessment of GPCR function for multiple, distinct members of the GPCRsuperfamily. The present methods may additionally be utilized instudying and understanding the mechanisms of actions of varioustherapeutic agents. The present inventors have determined that a proteinconjugate or chimera comprising an arrestin molecule and a detectablemolecule (such as Green Fluorescent Protein) is useful in such methodsof assaying in vivo GPCR activity).

Due to the therapeutic importance of GPCRs, methods for the rapidscreening of compounds for GPCR ligand activity are desirable.Additionally, methods of screening orphan GPCRs for interactions withknown and putative GPCR ligands assist in characterizing such receptors.Optical methods are available for studying labelled protein dynamics inintact cells, including video microscopy, fluorescence recovery afterphotobleaching, and resonance energy transfer. However, such methods areof limited usefulness in labeling GPCRs for study, due to the relativelylow level of GPCR expression and the alterations in receptor functionthat can occur after tagging or labeling of the receptor protein.Radiolabeling or fluorescent labeling of test ligands has also beenutilized in screening for GPCR ligands. See, e.g., Atlas et al., Proc.Natl. Acad. Sci. USA 74:5490 (1977); U.S. Pat. No. 5, 576,436 to McCabeet al. (all patents cited herein are incorporated herein in theirentirety). The introduction of foreign epitopes into receptor cDNA toproduce hybrid GPCRs is now a standard technique, and enhances detectionof GPCRs by monoclonal antibody technology. However, such techniques arelimited in their applicability to living cells. U.S. Pat. No. 5,284,746to Sledziewski describes yeast-mammalian hybrid GPCRs and methods ofscreening for GPCR ligands using such hybrid receptors. U.S. Pat. No.5,482,835 to King et al. describes methods of testing in yeast cells forligands of mammalian GPCRs. However, application of these techniques tothe study or identification of orphan GPCRs requires prior knowledge ofligands or signal transduction events and are therefor not generallyapplicable or universal.

Phosphorylation of GPCRs is a mechanism leading to desensitization ofthe receptors; receptors that have been continuously or repeatedlystimulated lose responsiveness, whereas the responses of other receptorsremain intact. See Harden, Pharmacol. Rev. 35:5 (1983); Benovic et al.,Annu. Rev. Cell. Biol. 4:405(1988). In a variety of cells, specifickinases have evolved for specific GPCRs. Desensitization occurs via thefollowing pathway: agonist occupancy of the receptor transforms thereceptor into an appropriate substrate for an associated kinase;β-arrestin binds to the kinase phosphorylated receptor and preventssubsequent interaction with the appropriate G-protein, as well asinitiating both internalization and resensitization processes. Fergusonet al, Science, 271:363 (1996); Lohse et al., Science 248: 1547 (1990).β-arrestin dependent desensitization is induced only when the GPCR isactivated by ligand desensitizes only its target receptors). Lohse etal., (1990) and Attramadal et al., J. Biol. Chem. 267:17882 (1992)provide cDNA and amino acid sequences of β-arrestin. Various isoforms ofβ-arrestin are known; as used herein, β-arrestin refers to all suchisoforms of β-arrestin, proteins having substantial sequence similaritythereto which are functional β-arrestins, and functional fragmentsthereof. Functional fragments of β-arrestin, its isoforms and analogs,may be determined using techniques as known in the art.

Molecules detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical and optical means are known. Opticallydetectable molecules include fluorescent labels, such as commerciallyavailable fluorescein and Texas Red. Detectable molecules useful in thepresent invention include any biologically compatible molecule which maybe conjugated to a β-arrestin protein without compromising the abilityof β-arrestin to interact with the GPCR system, and without compromisingthe ability of the detectable molecule to be detected. Conjugatedmolecules (or conjugates) of β-arrestin and detectable molecules (whichalso may be termed `detectably labelled β-arrestins`) are thus useful inthe present invention. Preferred are detectable molecules capable ofbeing synthesized in the cell to be studied (e.g., where the cell can betransformed with heterologous DNA so that the βarrestin-detectablemolecule chimera is produced within the cell). Particularly preferredare those detectable molecules which are inherently fluorescent in vivo.Suitable detectable molecules must be able to be detected withsufficient resolution within a cell that translocation of β-arrestinfrom the cytosol to the cell membrane in response to agonist binding toGPCR can be qualitatively or quantitatively assessed. Moleculesdetectable by optical means are presently preferred.

Fusion proteins with coding sequences for beta-galactosidase, fireflyluciferase, and bacterial luciferase have been used in methods ofdetecting gene expression and protein interactions in cells. However,these methods require exogenously-added substrates or cofactors. In themethods of the present invention, an inherently fluorescent markermolecule is preferred, such as GFP, since detection of such a markerintracellularly requires only the radiation by the appropriatewavelength of light and is not substrate limited.

Green Fluorescent Protein (GFP) was first isolated from the jelly fishAequorea victoria, and has an inherent green bioluminescence that can beexcited optically by blue light or nonradiative energy transfer.Sequences of GFP-encoding cDNA and GFP proteins are known; see, e.g.,Prasher et al., Gene, 111:229 (1992). The crystalline structure of GFPis described in Ormo et al., Science 273:1392 (1996). Purified nativeGFP absorbs blue light (maximally at 395 nm with a minor peak at 470 m )and emits green light (peak emission at 509 nm) (Morise et al,Biochemistry, 13:2656 (1974); Ward et al., Photochem. Photobiol., 31:611(1980)). It has been shown that GFP expressed in prokaryotic andeukaryotic cells produces a strong green fluorescence when excited bynear UV or blue light (see U.S. Pat. No. 5, 491,084 to Chalfie andPrasher); as this fluorescence requires no additional gene products fromA. victoria, chromophore formation is not species specific and occurseither through the uses of ubiquitous cellular components or byautocatalysis. Expression of GTP in Escherichia coli results in aneasily detected green fluorescence that is not seen in control bacteria.See Chalfie et al., Science 263:802 (1994); U.S. Pat. No. 5,491,084.Cells expressing the green-fluorescent proteins may be convenientlyseparated from those which do not express the protein by afluorescence-activated cell sorter.

As used herein, Green Fluorescent Protein refers to the variousnaturally occurring forms of GFP which can be isolated from naturalsources, as well as artificially modified GFPs which retain thefluorescent abilities of native GFP. As discussed in Ormo et al.,Science 273:1392 (1996), various mutants of GFP have been created withaltered excitation and emission maxima. Two characteristics of wild-typeGFP which affect its usefulness in mammalian cell lines are the need toexcite it at UV wavelengths to obtain a maximal fluorescent signal, anddecreased fluorescence at temperatures over 23° C. However, the S65T/GFPmutant overcomes these limitations. Heim et al., Proc. Natl. Acad. Sci.USA91:12501 (1994). Additional alterations in the GFP protein sequencewhich provide inherently fluorescent, biologically compatible moleculeswill be apparent to those in the art; sequence alterations may be madeto alter the solubility characteristics of the protein, its excitationwavelength, or other characteristics, while retaining useful fluorescentproperties. See, e.g. U.S. Pat. No. 5,625,048 to Tsien and Heim; WO9711091 (Bjorn, Poulsen, Thastrup and Tullin); WO 9627675 (Haseloff,Hodge, Prasher and Siemering); WO 9627027 (Ward); WO 9623898 (Bjorn etal.); WO 9623810 (Heim and Tsien); WO 9521191 (Chalfie and Ward).

Cells useful in the methods of the present invention include eukaryoticand prokaryotic cells, including but not limited to bacterial cells,yeast cells, fungal cells, insect cells, nematode cells, plant or animalcells. Suitable animal cells include, but are not limited to HEK cells,HeLa cells, COS cells, and various primary mammalian cells. Cellscontained in intact animals, including but not limited to nematodes,zebrafish (and other transparent or semi-transparent animals) andfruitflies, may also be used in the methods of the present invention. Ananimal model expressing a βarrestin-detectable molecule fusion proteinthroughout its tissues, or within a particular organ or tissue type,will be useful in studying cellular targets of known or unknown GPCRligands.

Cells useful in the present methods include those which express a knownGPCR or a variety of known GPCRs, or which express an unknown GPCR or avariety of unknown GPCRs. As used herein, a cell which expresses a GPCRis one which contains that GPCR as a functional receptor in itsmembrane; the cells may naturally express the GPCR(s) of interest, ormay be genetically engineered to express the GPCR(s) of interest. Asused herein, an `unknown` or `orphan` receptor is one whose function isunknown, and/or whose ligands are unknown.

The present experiments

Green fluorescent protein (GFP) has been used to study protein--proteininteractions in living cells. See Kaether & Gerdes, FEBS Lett. 369:267(1995); Olson et al., J. Cell. Biol. 130:639 (1995). Green fluorescentprotein (GFP) is useful as a reporter molecule for fusion proteins dueto its inherent fluorescence and its folding, which apparently isolatesit from its conjugated partner. Prasher et al., Gene 111:229 (1992);Ormo et al., Science 273:1392 (1996). For example, a seven transmembraneprotein as complex as the β2AR, which is three times larger than GFP,exhibits normal biochemistry after GFP conjugation to its C-terminus.Barak et al., Mol. Pharmacol. 51:177 (1997).

The present inventors established that a fusion protein consisting of aβ-arrestin molecule (β-arrestin2) conjugated to a GFP at its C-terminus(βarr2-GFP, FIG. 1) is expressed in cells and is biologically active.The βarr2-GFP fusion protein is approximately 50% larger thanβ-arrestin2, and this size increase is reflected by its slower migrationon SDS-Page (FIG 2A). The left lane of FIG. 2A, exposed to an antibodyagainst β-arrestin, shows that βarr2-GFP runs more slowly thanendogenous β-arrestin2 (highlighted middle band). The right lane of FIG2A, treated with a monoclonal anti-GFP antibody, demonstrates that theslower band does indeed contain GFP.

β2AR normally sequesters poorly in COS cells, and this has beencorrelated to the relatively poor expression of endogenous β-arrestinsin COS cells. Menard et al., Mol. Pharmacol. 51:800 (1997); Zhang etal., J. Biol. Chem. 271:18302 (1996). Overexpression of exogenousβ-arrestin enhances β2AR sequestration in these cells; similarly, asshown herein, βarr2-GFP overexpression in COS cells augmented β2ARinternalization (FIG. 2B), demonstrating that βarr2-GFP is biologicallyactive and equivalent to native β-arrestin.

Biochemical evidence indicates that β-arrestins are predominantlycytosolic proteins. Ferguson et al., Can. J. Physiol. Pharmacol. 74:1095(1996). The present inventors, using confocal microscopy of βarr2-GFP inHEK-293 cells (FIG. 3A, left panel), confirmed that βarr2-GFP isdistributed throughout the cytosol and excluded from the nucleus. Thepresent data also establish for the first time that β-arrestin is notpredominantly compartmentalized at the plasma membrane in the absence ofagonist but that, upon addition of saturating concentrations of anagonist to the cell medium, β-arrestin is translocated from cell cytosolto cell membrane. Where β-arrestin is conjugated to an opticallydetectable molecule such as GFP, as shown herein, a rapid and readilyobservable optical enhancement of the membrane and a concomitant loss ofcytosolic optically signals occurs (see FIGS. 3A and 3B, where membranefluorescence is enhanced and cytosol fluorescence is decreased due totranslocation of the βarrestin-GFP chimera).

To investigate whether the intracellular translocation of β-arrestintargeted binding sites in the plasma membrane other than the β2AR, thepresent inventors first crosslinked the receptors using monoclonalantibodies. As reported herein and shown in FIG. 4, the geometry of theagonist-induced time dependent translocation of β-arrestin to the plasmamembrane mimicked the distribution of pre-aggregated β2ARs, indicatingthat the targeted site of β-arrestin is indeed β2AR or an associatedcomponent.

It has been postulated that phosphorylation of GPCRs by GRKs facilitatesdesensitization by increasing their affinity for β-arrestins. Gurevichet al, J. Biol. Chem. 268:16879 (1993); Gurevich et al., J. Biol. Chem.268:11628 (1993). When expressed in HEK-293 cells and exposed toagonist, mutant Y326A-β2ARs are not significantly phosphorylated byendogenous GRKs (Ferguson et al., J. Biol. Chem., 270:24782 (1995).Therefore, the present inventors utilized this mutant receptor toinvestigate the above question of β-arrestin affinity in vivo.Y326A-β2AR was cotransfected with βarr2-GFP into HEK cells in theabsence and presence of co-transfected GRK. If the above hypothesis weretrue, reversal of phosphorylation impairment by overexpressed GRKs wouldresult in a noticeable difference in βarr2-GFP translocation. Asreported herein, without added GRK, βarr2-GFP translocation in responseto agonist proceeded poorly; with the addition of GRK, βarr2-GFPtranslocation to the plasma membrane was much more robust (FIG. 5),indicating the importance of phosphorylation to β-arrestin activity.

The present inventors determined that translocation of β-arrestin fromthe cell cytosol to the cell membrane is an indicator of agoniststimulation of GPCR activity, and that a chimeric protein comprisingβ-arrestin and the detectable molecule GFP was capable of detectablydisplaying the real-time translocation of β-arrestin in response toagonist activation of GPCRs.

The results presented herein establish that β-arrestin targets GPCRs oran associated molecule following agonist binding and receptorphosphorylation. These data demonstrate a biological behavior forβ-arrestin that has only been postulated from biochemical studies, andcharacterize for the first time how β-arrestin compartmentalizationchanges after initiation of receptor signal transduction. Agonistactivation of a GPCR ultimately culminates in the association ofβ-arrestins with GPCRs, thus the visualization of the agonist mediatedβ-arrestin translocation process provides a universal indicator of GPCRactivation.

The present inventors have demonstrated that GPCR signal transductioninduces a rapid, substantial increase in the relative and absoluteamount of plasma membrane bound β-arrestin. The agonist-mediatedredistribution of β-arrestin coupled to a detectable molecule providesan optical amplification of the extracellular signals transduced byGPCRs, and this occurs simultaneous with, or within the same time frameas, the chemical amplification normally provided by second messengercascades. Chimeras of β-arrestin and a detectable molecule are usefulfor the study of β-arrestin kinetics and GPCR related behavior such asendocytosis. Additionally, such chimeras are useful as biosensors forsignaling when GPCRs become activated, and provide methods of screeningcompounds for GPCR activity, and screening orphan GPCRs for ligandresponsiveness. In addition, the ability of co-transfected GRKs toenhance both the rate and extend of β-arrestin translocation indicatethat the present methods and constructs can also be used to monitor GRKactivity, as well as monitor drugs, proteins and compounds foractivation or inhibition of the GRK/β-arrestin process.

The present invention provides a method for screening compounds for GPCRagonist activity, comprising: a) providing a cell expressing a known orunknown GPCR and containing a chimeric protein comprising a β-arrestinprotein and a visually detectable protein; b) exposing the cell to atest compound; and c) detecting translocation of the detectable moleculefrom the cytosol of the cell to the membrane edge of the cell; wheretranslocation of the detectable molecule from the cytosol to themembrane edge of the cell indicates activation of the GPCR and,accordingly, the GPCR activating effect of the test compound.Translocation of the chimeric protein is evidenced by an increase in theintensity of detectable signal located at the membrane edge (and/or adecrease in the cytosol), where the change occurs after exposure to thetest compound. Translocation may thus be detected by comparing changesin the detectable signal in the same cell over time (i.e., pre and posttest compound exposure). Alternatively, a test cell may be compared to acontrol cell (no exposure to test compound), or a test cell may becompared to a pre-established standard. If a known agonist is availablethe present methods can be used to screen for and study GPCRantagonists. Additionally, the membrane association of β-arrestin shouldbe increased by expression of an excess of receptor or by aconstitutively active GPCR that undergoes phosphorylation by GRKs evenin the absence of agonist. Therefore, the present methods can be used tomonitor for inverse agonists of GPCRs.

Methods of detecting the intracellular translocation of the chimericprotein will depend on the particular detectable protein utilized; oneskilled in the art will be able to readily devise detection methodssuitable for particular detectable molecules, given the teachings of thepresent specification and knowledge in the art. In a preferredembodiment, the visually detectable protein is a green-fluorescentprotein (GFP) as discussed below.

The methods of the present invention provide easily detectable results.The translocation of β-arrestin coupled to a detectable molecule such asGFP, in response to GPCR activation, results in a relative enhancementof the detectable signal at the cell edge (i.e., at the cell membrane).In addition, the concomitant decrease in detectable signal from the cellcytosol means that `background noise` (detectable signals which do notchange in response to GPCR activation) is minimized. In certain cells,activation of GPCRs will result in essential clearing of detectablesignal from the cytosol, and a 100-fold increase (or more) in thedetectable signal at the cell membrane. In the present methods, it ispreferred that the detectable signal at the membrane edge increase,after GPCR activation, at least two-fold, more preferably at least3-fold, and more preferably at least 5-fold or at least ten-fold.

As used herein, the introduction of a chimeric protein into a cell maybe accomplished by introducing into the cell (or the cell's ancestor) anucleic acid (e.g., DNA or RNA) sequence or construct encoding thechimeric protein, and culturing the cell in an environment which allowsexpression of the chimeric protein. Introduction of nucleic acidsencoding the chimeric protein, or introduction of the protein itself,into a cell may be carried out by any of the many suitable methods whichare known in the art, including transfection, electroporation,microinjection, and liposome delivery.

The present invention provides a DNA construct comprising a promoter,DNA encoding a β-arrestin protein operatively associated therewith, andDNA encoding a visually detectable marker protein operatively associatedtherewith. The promoter is operatively associated with the encoding DNA;DNA encoding β-arrestin may be 5' from DNA encoding the visuallydetectable marker, or vice versa. In a preferred embodiment, the NDAencoding a visually detectable marker encodes a green-fluorescentprotein (GFP). Vectors comprising such DNA constructs are a furtheraspect of the present invention.

The present invention further provides conjugates (such a chimericproteins or fusion proteins) which comprise a β-arrestin protein and avisually detectable protein. In a preferred embodiment, the visuallydetectable protein is a green-fluorescent protein (GFP).

The present invention further provides a cell comprising a DNA molecule,which DNA molecule comprises, in the 5' to 3' direction, a promoter, DNAencoding a β-arrestin protein operatively associated therewith, and DNAencoding a visually detectable marker protein operatively associatedtherewith. In a preferred embodiment, the DNA encoding a visuallydetectable marker encodes a green-fluorescent protein (GFP).

The cells of the present invention may be used to detect the presence ofspecific molecules in various kinds of samples such as, e.g., aqueoussamples, biological samples (for example blood, urine or saliva),environmental samples, or industrial samples. In such uses, the cellscontain a GPCR whose agonists are known. Activation of the GPCR and theconcomitant translocation of the detectable signal from the cytosol tothe membrane edge indicates the presence of the agonist for the GPCR. Acell used in such a method may contain only a single type of known GPCR,or a variety of known GPCRs. Such detection will be useful for medicaland veterinary diagnostic purposes; industrial purposes; and screeningfor drugs or chemicals of abuse or biological toxins that affectGPCR-mediated signal transduction.

The cells of the present invention may be deposited on, affixed to,supported by, or immobilized on a substrate. The substrate may be of anysuitable material which is not harmful or detrimental to the livingcells deposited thereon, i.e., which is bio-compatible with the livingmaterial deposited thereon. The substrate may be rigid, semi-rigid orflexible; and may be opaque, transparent, or semi-transparent. The size,geometry and other physical characteristics of the substrate will bedictated by the intended use, as will be apparent to one skilled in theart. Suitable substrates include, but are not limited to, plastics,glass, ceramics, silica, biocompatible monomer and polymer compositions,semiconductor materials, fiber optic materials, polystyrene, membranes,sephadex, and bio-organic materials. Examples of biocompatible materialsare provided in U.S. Pat. Nos. 5,578,079; 5,575,997 and 5,582,834 toLeung and Clark; and 5,522,896 to Prescott.

The present invention further provides methods for screening for thepresence of a GPCR agonist in a solution which comprises: a) providing acell expressing a known or unknown GPCR and containing a chimericprotein comprising a β-arrestin protein and a visually detectableprotein; b) exposing the cell to a test solution; and c) detectingtranslocation of the detectable molecule from the cytosol of the cell tothe membrane edge of the cell; where translocation of the detectablemolecule from the cytosol to the membrane edge of the cell indicatesactivation of the GPCR and, accordingly, the GPCR agonist effect of thetest solution. Translocation of the chimeric protein is evidenced asdiscussed above.

The present invention further provides methods for screening for thepresence of a GPCR antagonist in a solution which comprises: a)providing a cell expressing a GPCR and containing a chimeric proteincomprising a β-arrestin protein and a visually detectable protein; b)exposing the cell to a test compound; then c) exposing the cell to aknown agonist to the GPCR expressed in the cell; and d) detectingtranslocation of the detectable molecule from the cytosol of the cell tothe membrane edge of the cell. If the test compound contains anantagonist, translocation of the detectable molecule will be delayed fora period of time corresponding to duration of antagonist action on thereceptor (which time period will vary depending on the antagonist and/orthe receptor). Translocation of the detectable molecule from the cytosolto the membrane edge of the cell indicates activation of the GPCR by theagonist. Accordingly, when translocation does not occur or is delayed(Compared to that which would occur in the absence of test compound),the test compound contains an antagonist to the GPCR. Absence or delayof translocation may be assessed by comparison to a control cell (notexposed to test compound) or to a predetermined standard. Translocationof the chimeric protein is evidenced as discussed above. Exposure to thetest compound and the known agonist may occur at essentially the sametime, or exposure to the agonist may occur subsequent to exposure to thetest compound. As used herein, subsequent exposure refers to exposurewithin the time period during which a potential antagonist would beexpected to be interacting with the GPCR (i.e., binding to or bound tothe GPCR).

The present invention further provides methods for screening a cell forthe presence of a GPCR, comprising: a) providing a test cell; b)introducing into the test cell a chimeric protein comprising aβ-arrestin protein and a visually detectable protein; and then c)exposing the cell to a test solution containing a known agonist to aGPCR; and d) detecting translocation of the detectable molecule from thecytosol of the cell to the membrane edge of the cell; wheretranslocation of the detectable molecule from the cytosol to themembrane edge of the cell indicates activation of a GPCR and,accordingly, that the test cell contains such a GPCR. Translocation ofthe chimeric protein is evidenced as discussed above.

The present invention further provides methods for screening a cellpopulation for the presence of cells containing GPCRs, comprising: a)providing a population of test cells, said test cells containingchimeric proteins comprising a β-arrestin protein and a visuallydetectable protein; and then b) exposing the cell population to a testsolution containing an agonist to a GPCR; and d) detecting those cellsin which translocation of the detectable molecule from the cytosol ofthe cell to the membrane edge of the cell occurs; where translocation ofthe detectable molecule from the cytosol to the membrane edge of a cellindicates activation of a GPCR and, accordingly, that the cell inquestion contains a GPCR. Translocation of the chimeric protein isevidenced as discussed above. Populations of cells to be screenedinclude a collection of individuals cells, a tissue comprising aplurality of similar cells, an organ comprising a plurality of relatedcells, or an organism comprising a plurality of tissues and organs.

As used herein, `exposing` a cell to a test compound or solution meansbrining the cell exterior in contact with the test compound or solution.Where the test compound or solution is being screened for GPCR ligandactivity, exposure is carried out under conditions that would permitbinding of GCPR ligand to a receptor expressed in that cell. As usedherein, `translocation` of β-arrestin refers to movement of theβ-arrestin molecule from one area of the cell to another.

The present methods may further be used to assess or study the effectsof any molecule in the GPCR pathway which exerts its effect upstream ofβ-arrestin binding (i.e., prior to β-arrestin binding to thephosphorylated GPCR). Thus the present invention provides methods forassessing GPCR pathway functions in general. As used herein, the GPCRpathway refers to the series of events which starts with agonistactivation of a GPCR followed by desensitization of the receptor via Gprotein-coupled receptor kinase (GRK) phosphorylation and β-arrestinbinding.

In a broad sense the present invention thus provides a method ofscreening test compounds and test conditions for the ability to affect(activate or inhibit, enhance or depress) a GPCR pathway, and providesmethods of assessing GPCR pathway function in a cell in general. In thepresent methods, the extent of translocation of β-arrestin is indicatedby the degree of detectable changes in the cell; the extend ofβ-arrestin translocation is an indicator under varied test conditionsmay be compared, or a test condition may be compared to a controlcondition or to a predetermined standard.

For example, the specificity and effects of various kinases (includingthose know to interact with GPCR pathways and those not previously knownto interact with GPCRs) for a specific GPCR or a group of GPCRs may beassessed by providing a test kinase to a test cell expressing a GPCR andcontaining a detectable β-arrestin molecule, exposing the cell to a GPCRagonist, and assessing the translocation of detectable β-arrestin fromthe cell cytosol to the cell membrane (see Example 7 herein).Translocation of the β-arrestin to the cell membrane indicates that thetest kinase, in response to agonist occupancy of the receptor, is ableto bind to and phosphorylate the receptor, so that β-arrestin will thenbind to the kinase phosphorylated receptor and prevent subsequentinteraction with the appropriate G-protein. In similar ways, thefunction of altered, recombinant or mutant kinases may be assessed;compounds may be screened for the ability to activate or inhibit theGPCR pathway, G protein-coupled receptor kinases, or β-arrestin binding;and the function of G-proteins may be assessed. For example, thefollowing test conditions may be assessed using methods as describedherein: the effects of G-proteins (including natural, heterologous, orartificially altered G-proteins) within the test cell; exposure of thetest cell to known or putative GPCR ligands; and co-expression of asecond receptor in the test cell expressing a GPCR.

Still further, the present methods allow the screening of β-arrestins(naturally occurring, artifically introduced or altered, mutant orrecombinant) for the ability to bind to a phosphorylated GPCR. In suchmethods, the test β-arrestin is conjugated to a detectable molecule suchas GFP, and is placed within a cell containing a GPCR. The cell isexposed to a known agonist of the GPCR, and translocation of thedetectable molecule from the cytosol of the cell to the membrane edge ofthe cell is detected. The translocation of the detectable moleculeindicates that the test β-arrestin protein is able to bind to thephosphorylated GPCR. As in other methods of the present invention, thetranslocation may be compared to a control cell containing a knownβ-arrestin or to a predetermined standard.

G. Protein Coupled Receptors

GPCRs suitable for use in the present methods are those in which agonistbinding induces G protein-coupled receptor kinase (GRK) phosphorylation;translocation of arrestin from the cytosol of the cell to the cellmembrane subsequently occurs. As it is believed that virtually allmembers of the GPCR superfamily desensitize via this common mechanism,examples of suitable types of GPCRs include but are not limited to betaand alpha adrenergic receptors; GPCR binding neurotransmitters (such asdopamine); GPCRs binding hormones; the class of odorant receptors(taste, smell and chemotactic receptors as found in nasal mucosa and thetongue, and on sperm, egg, immune system cells and blood cells); theclass of type II GPCRs including secretin, glucagon, and other digestivetract receptors; light-activated GPCRs (such as rhodopsin); and membersof the type III family of GPCRs which include but are not limited tometabotopic glutamate receptors and GABA_(B) receptors. In addition tonaturally occurring GPCRs, GPCRs may be specifically engineered orcreated by random mutagenesis. Such non-naturally occurring GPCRs mayalso be utilized in and screened by the present methods. The presentmethods may be utilized with any membrane receptor protein in whichagonist binding results in the translocation of β-arrestin. Suchreceptors include growth factors that signal through G proteins.

Automated Screening Methods

The methods of the present invention may be automated to provideconvenient, real time, high volume methods of screening compounds forGPCR ligand activity, or screening for the presence of GPCR ligand in atest sample. Automated methods are designed to detect the change inconcentration of labelled β-arrestin at the cell membrane and/or in thecytosol after exposure to GPCR agonist. The alteration of β-arrestindistribution can be detected over time (i.e., comparing the same cellbefore and after exposure to a test sample), or by comparison to acontrol cell which is not exposed to the test sample, or by comparisonto pre-established indicia. Both qualitative assessments(positive/negative) and quantitative assessments (comparative degree oftranslocation) may be provided by the present automated methods, as willbe apparent to those skilled in the art.

It is thus a further object of the present invention to provide methodsand apparatus for automated screening of GPCR activity, by detecting thetranslocation of detectably labeled β-arrestin from cell cytosol to cellmembrane in response to agonist activation of GPCRs. The translocationmay be indicated by an alteration in the distribution of a detectablesignal within a cell over time, between a test cell and a control cell,or by comparison to previously established parameters. In particular,according to one embodiment of the present invention, a plurality ofcells expressing GPCRs and containing chimeric proteins comprising adetectable molecule and a β-arrestin molecule are provided. Indicia ofthe distribution of the detectable molecules are then measured usingconventional techniques. In various embodiments, (a) measurement ofoptical indicia occurs before and after the addition of a test sample toa cell, and the time point measurements are compared; (b) opticalindicia are measured in a test cell exposed to a test sample and in anon-exposed control cell, and these measurements are compared; and (c)measurement of a test cell after addition of a test sample is comparedto preestablished parameters. The optical indicia being measured may befluorescence signals (e.g., fluorescence intensities) if the detectablemolecule of the chimeric β-arrestin protein is a fluorescent indicatorsuch as GFP. Other optical indicia that are suitable for real-timemeasurement may also be used, as will be apparent to those skilled inthe art.

An embodiment of the present invention includes an apparatus fordetermining GPCR response to a test sample. This apparatus comprisesmeans, such as a fluorescence measurement tool, for measuring indicia ofthe intracellular distribution of detectable β-arrestin proteins in atleast one test cell, and optionally also in a control or calibrationcell. Measurement points may be over time, or among test and controlcells. A computer program product controls operation of the measuringmeans and performs numerical operations relating to the above-describedsteps. The preferred computer program product comprises a computerreadable storage medium having computer-readable program code meansembodied in the medium. Hardware suitable for use in such automatedapparatus will be apparent to those of skill in the art, and may includecomputer controllers, automated sample handlers, fluorescencemeasurement tools, printers and optical displays. The measurement toolmay contain one or more photodetectors for measuring the fluorescencesignals from samples where fluorescently detectable molecules areutilized in the detectable β-arrestin construct. The measurement toolmay also contain a computer-controlled stepper motor so that eachcontrol and/or test sample can be arranged as an array of samples andautomatically and repeatedly positioned opposite a photodetector duringthe step of measuring fluorescence intensity.

The measurement tool is preferably operatively coupled to a generalpurpose or application specific computer controller. The controllerpreferably comprises a computer program produce for controllingoperation of the measurement tool and performing numerical operationsrelating to the above-described steps. The controller may accept set-upand other related data via a file, disk input or data bus. A display andprinter may also be provided to visually display the operationsperformed by the controller. It will be understood by those having skillin the art that the functions performed by the controller may berealized in whole or in part as software modules running on a generalpurpose computer system. Alternatively, a dedicated stand-alone systemwith application specific integrated circuits for performing the abovedescribed functions and operations may be provided.

As provided above, the indicia of β-arrestin distribution may take theform of fluorescent signals, although those skilled in the art willappreciate that other indicia are known and may be used in the practiceof the present invention, such as may be provided by labels that producesignals detectable by fluorescence, radioactivity, colorimetry, X-raydiffraction or absorption or magnetism. Such labels include, forexample, fluorophores, chromophores, radioactive isotopes (e.g., ³² P or¹²⁵ I) and electron-dense reagents.

Definitions

As used herein, exogenous or heterologous DNA (or RNA) refers to DNA (orRNA) which has been introduced into a cell (or the cell's ancestor)through the efforts of humans. Such heterologous DNA may be a copy of asequence which is naturally found in the cell being transformed, or asequence which is not naturally found in the cell being transformed, orfragments thereof.

As used herein, the term `gene` refers to a DNA sequence thatincorporates (1) upstream (5') regulatory signals including a promoter,(2) a coding region specifying the product, protein or RNA of the gene,(3) downstream (3') regions including transcription termination andpolyadenylation signals and (4) associated sequences required forefficient and specific expression.

Use of the phrase "substantial sequence similarity" in the presentspecification refers to DNA, RNA or amino acid sequences which haveslight and non-consequential sequence variations from a sequence ofinterest, and are considered to be equivalent to the sequence ofinterest. In this regard, "slight and non-consequential sequencevariations" means that "similar" sequences (i.e., sequences that havesubstantial sequence similarity) will be functionally equivalent.Functionally equivalent sequences will function in substantially thesame manner to produce substantially the same compositions.

As used herein, a "native DNA sequence" or "natural DNA sequence" meansa DNA sequence which can be isolated from non-transgenic cells ortissue. Native DNA sequences are those which have not been artificiallyaltered, such as by site-directed mutagenesis. Once native DNA sequencesare identified, DNA molecules having native DNA sequences may bechemically synthesized or produced using recombinant DNA procedures asare known in the art.

As used herein, "a regulatory element" from a gene is the DNA sequencewhich is necessary for the expression of the gene, such as a promoter.In this invention, the term "operatively linked" to means that followingsuch a link a regulatory element can direct the expression of a linkedDNA sequence.

The term `promoter` refers to a region of a DNA sequence thatincorporates the necessary signals for the efficient expression of acoding sequence. This may include sequences to which an RNA polymerasebinds but is not limited to such sequences and may include regions towhich other regulatory proteins bind together with regions involved inthe control of protein translation and may include coding sequences.Suitable promoters will be apparent to those skilled in the art, andwill vary depending upon the cell in which the DNA is to be expressed. Asuitable promoter for use in DNA constructs encoding aβ-arrestin/detectable molecule construct may be a promoter naturallyfound in the cell in which expression is desired; optionally, thepromoter of the β-arrestin within the construct may be utilized. Bothinducible and constitutive promoters are contemplated for use in thepresent invention.

DNA Constructs

DNA constructs, or "expression cassettes," of the present inventioninclude, 5' to 3' in the direction of transcription, a promoter, a DNAsequence operatively associated with the promoter, and, optionally, atermination sequence including stop signal for RNA polymerase and apolyadenylation signal for polyadenylase. All of these regulatoryregions should be capable of operating in the cell to be transformed.Suitable termination signals for a given DNA construct will be apparentto those of skill in the art.

The term "operatively associated," as used herein, refers to DNAsequences on a single DNA molecule which are associated so that thefunction of one is affected by the other. Thus, a promoter isoperatively associated with a DNA when it is capable of affecting thetranscription of that DNA (i.e., the DNA is under the transcriptionalcontrol of the promoter). The promoter is said to be "upstream" from theDNA, which is in turn said to be "downstream" from the promoter.

The expression or transcription cassette may be provided in a DNAconstruct which also has at least one replication system. Forconvenience, it is common to have a replication system functional inEscherichia coli, such as Co/E1, pSC101, pACYC184, or the like. In thismanner, at each stage after each manipulation, the resulting constructmay be cloned, sequenced, and the correctness of the manipulationdetermined. In addition, or in place of the E. coli replication system,a broad host range replication system may be employed, such as thereplication systems of the P-1 incompatibility plasmids, e.g., pRK290.In addition to the replication system, there will frequently be at leastone marker present, which may be useful in one or more hosts, ordifferent markers for individual hosts. That is, one marker may beemployed for selection in a prokaryotic host, while another marker maybe employed for selection in a eukaryotic host. The markers may beprotection against a biocide, such as antibiotics, toxins, heavy metals,or the like; may provide complementation, by imparting prototrophy to anauxotrophic host; or may provide a visible phenotype through theproduction of a novel compound in the plant.

The various fragments comprising the various constructs, expressioncassettes, markers, and the like may be introduced consecutively byrestriction enzyme cleavage of an appropriate replication system, andinsertion of the particular construct or fragment into the availablesite. After ligation and cloning the DNA construct may be isolated forfurther manipulation. All of these techniques are amply exemplified inthe literature as exemplified by J. Sambrook et al., Molecular Cloning,A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).

The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof. As usedherein, βarr2-GFP=β-arrestin2 green fluorescent protein; GFP=greenfluorescent protein; GPCR=G protein-coupled receptor, βARK=betaadrenergic receptor kinase; GRK=G protein-coupled receptor kinase;β2AR=beta 2 adrenergic receptor; HEK-293=human embryonic kidney cells;DMEM=Dulbecco's modified Eagle medium; and MEM=Minimal Essential Medium.

EXAMPLE 1

Materials and Methods

Materials: Isoproterenol was obtained from Sigma RBI. Anti-mouseantibody was obtained from Sigma Chemicals or Molecular Probes. Mousemonoclonal antibody against the 12CA5 epitope was obtained fromBoehringer Mannheim. Cell culture media was obtained from Mediatech andfetal bovine serum from Atlanta Biologicals. Physiological buffers werefrom Gibco-Life Technologies Inc. Restriction enzymes were obtained fromPromega or New England Biolabs, T4 ligase was from Promega, and Hot TubDNA polymerase from Amersham. Commercially available plasmids containingvariants of Green Fluorescent Protein were obtained from Clontech.

Cell Culture and Transfection: HEK-293 and COS cells were maintained andtransfected as described by Barak et al., Mol. Pharm. 51:177 (1997).Cells containing both beta2 adrenergic receptor and β-arrestinconstructs were transfected with between 5-10 μg of receptor cDNA inpcDNA1/AMP and 0.5-1 μg of Barr2-GFP cDNA per 100 mm dish. GRKs wereexpressed using 5 μg of transfected cDNA in pcDNA1/AMP per dish.

Confocal Microscopy: HEK-293 cells transfected as described above wereplated onto 35 mm dishes containing a centered, 1 cm well formed from ahole in the plastic sealed by a glass coverslip. Primary and secondaryantibody labeling of live cells were performed at 37° C. for 30 minutesin media without serum in a 5% CO₂ incubator. Cells were washed threetimes between applications. Cells plated above in MEM or DMEM bufferedwith 20 mM Hepes were viewed on a Zeiss laser scanning confocalmicroscope.

Sequestration: Flow cytometry analysis was performed using techniquesknown in the art, as described in Barak et al., J. Biol. Chem. 269:2790(1994).

EXAMPLE 2

Construction of β-arrestin2-GFP Plasmid

β-arrestin2 cDNA in the plasmid pCMV5 was used as a template.Oligonucleotide primers surrounding a distal XhoI restriction site andthe C-terminal stop codon of β-arrestin2 were used to replace the stopcodon with an in frame BamHI restriction site by directed mutagenesis(Valette et al. Nucleic Acids Res. 17:723 (1989); Attramadal et al., J.Biol. Chem. 267:17882 (1992); Lohse et al., Science 248:1547 (1990)).The XhoI, BamHI segment was isolated. This segment was ligated to theN-terminal portion of β-arrestin cDNA (cut from pCMV5 by SacI and XhoI)in the polylinker of a plasmid that had been previously digested withScaI and BamHI and that contained S65T-Green Fluorescent Protein distaland in frame to the site of β-arrestin cDNA insertion. Lohse et al.,Science 248:1547 (1990). The resulting β-arrestin-GFP construct wasisolated following insertion and growth in E. coli. Constructs wereverified by sequencing.

A linear model of the β-arrestin2/S65T-GFP conjugate is provided in FIG.1.

EXAMPLE 3

Characterization of βarr2-GFP Expressed by HEK-293 Cells

Homogenates of HEK-293 cells transformed with the plasmid of Example 2were studied using known Western Blot techniques. The results showedthat HEK-293 cells expressed both endogenous β-arrestin and theβarr2-GFP conjugate.

Western blots of homogenates of HEK-293 cells transfected with theplasmid of Example 2 and expressing βarr2-GFP were performed. An equalamount of homogenate material was loaded into each of two lanes (FIG.2A). The left lane was exposed to anti-βarrestin antibody (Menard etal., Mol. Pharm. 51:800 (1997)), whereas the right lane was exposed to amouse monoclonal antibody against GFP. The βarr2-GFP fusion protein isapproximately 50% larger than β-arrestin2, and would thus be expected tomigrate more slowly than β-arrestin on SDS-Page.

Exposure to anti-βarrestin antibody revealed multiple bars (left lane);exposure to anti-GFP monoclonal antibody revealed a single bar (rightlane). The position of endogenous cellular β-arrestin2 is indicated bythe intermediate bar in the left lane (βarr2). The heavy band just below71,000 on the left lane (βarr2-GFP) is mirrored by a similar band in theright lane. In contrast, no band corresponding to endogenous cellularβ-arrestin 2 is observed with anti-GFP antibody exposure. The treatmentof the right lane with anti-GFP antibody demonstrated that the slowerband labelled by anti-βarrestin antibody contained GFP.

EXAMPLE 4

Biological Activity of βarrestin-GFP Conjugate

β-arrestin activity can indirectly be assessed by measuring its effecton receptor sequestration (see Menard et al., Mol. Pharm. 51:800 (1997);Ferguson et al., Science 271:363 (1996)). The β2AR normally sequesterspoorly in COS cells, and this has been correlated to the relatively poorexpression of endogenous β-arrestins (see Menard et al. Mol. Pharmocol.51:800 (1997); Ferguson et al., Science 271:363 (1996)). Overexpressionof exogenous β-arrestin enhances β2AR sequestration in these cells. Todemonstrate that the βarr2-GFP conjugate is a biologically activeβ-arrestin, COS cells overexpressing βarr2-GFP were examined foraugmentation of β2AR internalization, compared to the augmentation ofβAR2 seen with the overexpression of β-arrestin2. Results are shown inFIG. 2B.

Using epitope tagged βAR2 receptors, sequestration of βAR2 was studiedin COS cells overexpressing either (1) exogenous β-arrestin2 or (2) theβarr2-GFP conjugate. FIG. 2D shows the sequestration of β2AR in COScells with and without overexpressed β-arrestin2 (left two bars) andwith and without overexpressed βarr2-GFP (right two bars). Agonistmediated β2AR sequestration increased from 15±7% to 39±5% in thepresence of overexpressed β-arrestin2; overexpression of βarr2-GFPsimilarly increased agonist mediated β2AR sequestration from 25±4% to58±1%. Wild type β-arrestin2 and βarr2-GFP enhanced β2AR sequestrationequally well above control levels, producing a 2.5 and 2.4 fold increasein β2AR sequestration, respectively.

The above results indicated that the βarr2-GFP conjugate acts as abiologically active arrestin.

EXAMPLE 5

Agonist Mediated Translocation of βarr2-GFP

Agonist mediated translocation of the βarr2-GFP chimera from cellcytosol to membrane was studied using HEK-293 and COS cells transfectedwith plasmids containing cDNA for the β2AR receptor and for theβarr2-GFP conjugate.

HEK-293 and COS cells were transfected with plasmids containing 10 μg ofcDNA for β2AR and 0.5-1.0 μg for βarr2-GFP. Cells were assessed usingconfocal microscopy to detect the inherent intracellular fluorescence ofGFP.

Transfected HEK-293 cells are shown in FIG. 3A, where panel 1 depictscells prior to the addition of βAR2 agonist, and panel 2 depicts cellsfollowing the addition of agonist. Transfected COS cells are shown inFIG. 3B, where panel 1 depicts cells just prior to the addition of βAR2agonist, and panel 2 depicts cells ten minutes after the addition ofagonist.

As shown in FIG. 3A, βarr2-GFP distribution in HEK-239 cells wasinitially cytosolic (panel 1). No significant nuclear or membraneenhancement was apparent. Following the addition of the βAR2 agonistisoproterenol to the cell medium, the real-time agonist-mediatedredistribution of βarr2-GFP was viewed using confocal microscopy. Tenminutes after isoproterenol addition (saturating concentrations),enhancement of membrane fluorescence was see with a concomitant loss ofcytosolic fluorescence, indicating that the βarr2-GFP distribution hadshifted to the membrane (panel 2). These results establish that inHEK-293 cells containing the β2AR, βarr2-GFP expressed by the cell istranslocated from cytosol to membrane following the addition of a βAR2agonist. Exposure of the test cells to GPCR agonist enhanced membranebound fluorescence ten-fold over that seen prior to agonist exposure.

As shown in FIG. 3B, βarr2-GFP distribution in COS cells was initiallycytosolic (panel 1). No significant nuclear or membrane enhancement wasapparent. Following the addition of the βAR2 agonist isoproterenol tothe cell medium, the real-time agonist-mediated redistribution ofβarr2-GFP was viewed using confocal microscopy. Ten minutes afterisoproterenol addition (saturating concentrations), enhancement ofmembrane fluorescence was seen with a concomitant loss of cytosolicfluorescence, indicating that the βarr2-GFP distribution had shifted tothe membrane (panel 2). These results establish that in COS cellscontaining the β2AR, βarr2-GFP expressed by the cell is translocatedfrom cytosol to membrane following the addition of a βAR2 agonist.

Comparing FIGS. 3A and 3B shows that the fluorescent signal is reducedin COS cells as compared to HEK cells, reflecting the lower efficiencyof sequestration of the β2AR in COS cells. However, even in COS cellsthe shift of βarr2-GFP in COS cells from cytosol to membrane followingthe addition of βAR2 agonist is clearly discernible due to thefluorescence of the GFP moiety.

The above experiments with COS and HEK-293 cells were reproduced exceptthat the βAR2 antagonist propranolol was added to the cell medium. Usingconfocal microscopy to visually track βarr2-GFP in the cell in realtime, as above, indicated that no shift in βarr2-GFP from cytosol tomembrane occurred in response to a βAR2 antagonist. As shown in FIG. 6E,addition of an agonist (middle panel) resulted in translocation ofβarr2-GFP from cytosol to membrane; subsequent addition of an antagonist(right panel) reversed the translocation (compare to control, leftpanel).

Biochemical evidence indicates that β-arrestins are predominantlycytosolic proteins. Ferguson et al. Can. J. Physiol. Pharmacol. 74:1095(1996). The present results confirm that βarr2-GFP is distributedthroughout the cytosol and excluded from the nucleus. These data alsoestablish that βarr2-GFP is not predominantly compartmentalized at theplasma membrane in the absence of agonist, but that upon exposure to anagonist the cellular βarr2-GFP shifts to the membrane. The presentresults further indicate that the shift of the βarr2-GFP conjugate inresponse to the addition of a G protein coupled receptor agonist can bedetected optically as an enhancement of membrane fluorescence and/or aconcomitant loss of cytosolic fluorescence, and that this response israpidly observed.

EXAMPLE 6

Intracellular βarr2-GFP Targets Membrane Receptors

FIG. 4 shows the time course of βarr2-GFP redistribution to plasmamembrane 12CA5(HA) tagged β2AR in HEK-293 cells, as shown by confocalmicroscopy.

The present example demonstrates that β2ARs are the target ofintracellular βarr2-GFP conjugate proteins. HEK-293 cells containing12CA5(HA) tagged β2AR receptors were studied. The receptors in theHEK-293 cells were reorganized into plasma membrane clusters (Row A) bycrosslinking with a mouse monoclonal antibody directed against anN-terminal epitope, followed by Texas Red conjugated goat anti-mouseantibody. In FIG. 4, the three panes of Row A show the same HEK-293 cellwith βAR2 receptors reorganized into plasma membrane clusters.

HEK-293 cells were then exposed to agonist (isoproterenol added to cellmedium, as above); the three panels of Row B in FIG. 4 were takenconsecutively after agonist addition (left to right, at 0, 3 and 10minutes post agonist addition). The real-time redistribution ofβarr2-GFP to the receptors over a ten minute period is thus demonstratedby comparing the panels of Row A and Row B of FIG. 4. In FIG. 4, arrowsindicate areas of colocalization and the bar=10 microns.

FIG. 4 demonstrates that the geometry of the agonist-induced timedependent translocation of βarr2-GFP to the plasma membrane mimicked thedistribution of pre-aggregated β2ARs. This indicates that the primarysite targeted by β-arrestin is the β2AR or a closely associatedcomponent.

EXAMPLE 7

Intracellular βarr2-GFP Targets Membrane Receptors

It has been postulated that phosphorylation of GPCRs by GRKs facilitatesdesensitization by increasing the affinity for β-arrestins. Gurevich etal., J. Biol. Chem. 268:16879 (1993); Gurevich et al. J. Biol. Chem.268:11628-11638 (1993); Ferguson et al., Can. J. Physiol. Pharmacol.74:1095 (1996). When expressed in HEK-293 cells and exposed to agonist,mutant Y326A-β2ARs are not significantly phosphorylated by endogenousGFKs. Barak et al., Biochem. 34:15407 (1995) ; Ferguson et al., J. Biol.Chem. 270:24782 (1995). This phosphorylation impairment in Y326A-βAR2sis reversed by overexpression of GFKs in the same cell. Menard et al.,Biochem. 35:4155 (1996). The Y326A mutant receptor was used toinvestigate β-arrestin affinity in vivo; the effect of overexpressed GFKon the Y326A-B2AR interaction with βarr2-GFP was shown.

Y326A-β2AR and βarr2-GFP were co-transfected into HEK-239 cells, in theabsence and presence of co-transfected GRK. If phosphorylation of GPCRsby GRKs facilitates desensitization by increasing their affinity forβ-arrestins, then overexpression of GRK would result in a noticeabledifference in βarr2-GFP translocation.

FIG. 5 shows the influence of overexpressed GFK on the redistribution ofβarr2-GFP in HEK-293 cells expressing the Y326A phosphorylation impairedβ2AR. Cells without (Row A) and with (Row B) overexpressed GRKs wereexposed to agonist, and the real-time redistribution of βarr2-GFP wasobserved. Without added GRK, βarr2-GFP translocation in response toagonist proceeded poorly, as shown in Row A of FIG. 5. βarr2-GFPtranslocation in cells containing overexpressed GRK (Row B) was morerobust, indicating an increased affinity of βarr2-GFP for receptor andthe relationship of phosphorylation and β-arrestin activity.

EXAMPLE 8

Testing of Additional Receptors in the β2AR/rhodopsin Subfamily

Twelve different members of the β2AR/rhodopsin subfamily of GPCRs havebeen studied. Cells expressing a particular GPCR, and containingβarrestin-GFP chimeric proteins were exposed to known agonists for theGPCR being studied. In each case, an observable translocation of theβarrestin-GFP chimeric proteins from the cell cytosol to the cellmembrane was produced within minutes following addition of the GPCRagonist (data not shown).

What is claimed is:
 1. A method of detecting G protein coupled receptor(GPCR) pathway activity in a cell expressing at least one GPCR andcontaining β-arrestin protein conjugated to an optically detectablemolecule, said method comprising detecting translocation of thedetectable molecule from the cytosol of the cell to the membrane edge ofthe cell, wherein said translocation of the detectable moleculeindicates activation of the GPCR pathway;and wherein said detecting stepcomprises (i) detecting an increase in said detectable molecule at saidmembrane edge; (ii) detecting a decrease in said detectable molecule insaid cytosol; or (iii) detecting both an increase in said detectablemolecule at said membrane edge and detecting a decrease in saiddetectable molecule in said cytosol.
 2. A method according to claim 1wherein said detection is of an increase in the detectable signal at themembrane edge of the cell over time.
 3. A method according to claim 1wherein said detection is of a decrease in the detectable signal in thecytosol of the cell over time.
 4. A method according to claim 1 whereinsaid translocation is detected by comparing the distribution of thedetectable signal in a test cell to the distribution of the detectablesignal in a control cell.
 5. A method according to claim 1 wherein saiddetection of the detectable signal occurs over time.
 6. A methodaccording to claim 1 wherein said translocation is detected by comparingthe distribution of the detectable signal in a test cell to apre-established standard.
 7. A method according to claim 1 wherein saiddetectable molecule is photochemically detectable.
 8. A method accordingto claim 1 wherein said detectable molecule is biochemically detectable.9. A method according to claim 1 wherein said detectable molecule isimmunochemically detectable.
 10. A method according to claim 1 whereinsaid detectable molecule is spectroscopically detectable.
 11. A methodaccording to claim 1 wherein said cell is a mammalian cell.
 12. A methodaccording to claim 1, wherein the cell is selected from the groupconsisting of bacterial cells, yeast cells, fungal cells, plant cellsand animal cells.
 13. A method according to claim 1 wherein the cellexpresses a GPCR whose function is known.
 14. A method according toclaim 1 wherein the cell expresses a GPCR whose function is unknown. 15.A method according to claim 1 wherein the cell expresses an odorantGPCR.
 16. A method according to claim 1, wherein the cell expresses aβ-adrenergic GPCR.
 17. A method according to claim 1, wherein the cellendogenously expresses a GPCR.
 18. A method according to claim 1,wherein the cell has been transformed to express a GPCR not endogenouslyexpressed by such a cell.
 19. A method according to claim 1, wherein thecells are deposited on a substrate prior to detecting translocation ofthe detectable molecule from the cytosol to the membrane edge.
 20. Amethod according to claim 1 wherein said cell is contained in a tissue.21. A method according to claim 1 wherein said cell is contained in anorgan.
 22. A method according to claim 1 wherein the cell expresses ataste GPCR.
 23. A method according to claim 1 wherein the cell is aninsect cell.
 24. A method according to claim 1, wherein said detectingstep comprisesdetecting an increase in said detectable molecule at saidmembrane edge.
 25. A method according to claim 1, wherein said detectingstep comprisesdetecting a decrease in said detectable molecule in saidcytosol.
 26. A method according to claim 1, wherein said detecting stepcomprisesdetecting both an increase in said detectable molecule at saidmembrane edge and detecting a decrease in said detectable molecule insaid cytosol.